Bottom Line:
Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy.Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues.This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression.

Affiliation: Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany.

ABSTRACTTargeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.

Mentions:
Reporter gene vectors were made carrying the PRSTSDP and PRSADLA peptides or controls and gene transduction in vivo was evaluated. In a first step, we investigated whether the selected AAV capsid variants home to lung tissue more efficiently than AAV control vectors (wild-type or random insert capsids). Vectors were administered intravenously, and DNA was recovered from lung tissue after 8 days. Quantitative PCR of the CMV promoter region of the vectors revealed an up to 63-fold higher yield for the selected capsid variants compared to AAV-2 wild-type vectors and up to 74-fold higher yield compared to random control insert vectors (Figure 5A). Evaluation of luciferase expression in the lung 28 days after intravenous administration revealed a 35-fold and 233-fold increased transduction efficiency of PRSADLA and PRSTSDP, respectively, compared to wild-type AAV (Figure 5B). To determine the specificity of lung-targeted capsids, luciferase expression in several control organs was evaluated. Both selected clones showed higher gene transduction in liver, heart, kidney, brain, and muscle, compared to unselected controls (Figure 5C), suggesting that the cellular target bound by the selected vectors in vivo is ubiquitously rather than lung-specifically expressed.

Mentions:
Reporter gene vectors were made carrying the PRSTSDP and PRSADLA peptides or controls and gene transduction in vivo was evaluated. In a first step, we investigated whether the selected AAV capsid variants home to lung tissue more efficiently than AAV control vectors (wild-type or random insert capsids). Vectors were administered intravenously, and DNA was recovered from lung tissue after 8 days. Quantitative PCR of the CMV promoter region of the vectors revealed an up to 63-fold higher yield for the selected capsid variants compared to AAV-2 wild-type vectors and up to 74-fold higher yield compared to random control insert vectors (Figure 5A). Evaluation of luciferase expression in the lung 28 days after intravenous administration revealed a 35-fold and 233-fold increased transduction efficiency of PRSADLA and PRSTSDP, respectively, compared to wild-type AAV (Figure 5B). To determine the specificity of lung-targeted capsids, luciferase expression in several control organs was evaluated. Both selected clones showed higher gene transduction in liver, heart, kidney, brain, and muscle, compared to unselected controls (Figure 5C), suggesting that the cellular target bound by the selected vectors in vivo is ubiquitously rather than lung-specifically expressed.

Bottom Line:
Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy.Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues.This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression.

Affiliation:
Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany.

ABSTRACTTargeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.